Selectively Modified Lactose and N-Acetyllactosamine Analogs at Three Key Positions to Afford Effective Galectin-3 Ligands

Galectins constitute a family of galactose-binding lectins overly expressed in the tumor microenvironment as well as in innate and adaptive immune cells, in inflammatory diseases. Lactose ((β-D-galactopyranosyl)-(1→4)-β-D-glucopyranose, Lac) and N-Acetyllactosamine (2-acetamido-2-deoxy-4-O-β-D-galactopyranosyl-D-glucopyranose, LacNAc) have been widely exploited as ligands for a wide range of galectins, sometimes with modest selectivity. Even though several chemical modifications at single positions of the sugar rings have been applied to these ligands, very few examples combined the simultaneous modifications at key positions known to increase both affinity and selectivity. We report herein combined modifications at the anomeric position, C-2, and O-3′ of each of the two sugars, resulting in a 3′-O-sulfated LacNAc analog having a Kd of 14.7 µM against human Gal-3 as measured by isothermal titration calorimetry (ITC). This represents a six-fold increase in affinity when compared to methyl β-D-lactoside having a Kd of 91 µM. The three best compounds contained sulfate groups at the O-3′ position of the galactoside moieties, which were perfectly in line with the observed highly cationic character of the human Gal-3 binding site shown by the co-crystal of one of the best candidates of the LacNAc series.


Introduction
Galectins are evolutionarily conserved, soluble glycan-binding proteins (lectins) found in a wide variety of taxonomic groups. Galectin-3 (Gal-3) is an exceptional member of the galectin family because of its unique structure and functional diversity. Unlike other galectins, Gal-3 is a chimeric protein composed of distinct domains. In human Gal-3, the C-terminal carbohydrate recognition domain (CRD) is connected to an N-terminal tail (NT). The NT possesses nine non-triple-helical collagen-like Pro/Gly-rich repeats followed by a 21-amino-acid-long N-terminal segment [1]. While the CRD contains the glycan-binding site, the non-lectin NT domain contributes to the oligomerization of this lectin [2].
The syntheses of the remaining propargylated LacNAc-based derivatives, having anionic groups at the key 3 -position, are illustrated in Scheme 4. Hence, bis-6,6 -O-silyl LacNAc intermediate 28 was first sulfated at the 3 position following the above tin acetal procedure (Bu 2 SnO, MeOH, 80 • C, 4 h) followed by sulfation using the Et 3 N-SO 3 complex (DMF, 60 • C, 17 h, 90% overall), which gave 29, which after silyl group deprotection as before, provided target compound 30 in 64% yield. To fully exploit the highly cationic character of the hGal-3 deep binding pocket, a carboxylate derivative was also prepared. In this case, compound 28 was treated under the tin acetal conditions described above but using tert-butyl bromoacetate as electrophile which afforded ester 31 in 80% yield. Acid deprotection of the t-butyl ester (TFA, DCM, r.t., 0.5 h) followed by desilylation and neutral-ization gave 32 (85%). The synthesis of the sialylated trisaccharide 33 ( Figures S43 and S44) has been recently described [32]. procedure (Bu2SnO, MeOH, 80 °C, 4 h) followed by sulfation using the Et3N-SO3 complex (DMF, 60 °C, 17 h, 90% overall), which gave 29, which after silyl group deprotection as before, provided target compound 30 in 64% yield. To fully exploit the highly cationic character of the hGal-3 deep binding pocket, a carboxylate derivative was also prepared. In this case, compound 28 was treated under the tin acetal conditions described above but using tert-butyl bromoacetate as electrophile which afforded ester 31 in 80% yield. Acid deprotection of the t-butyl ester (TFA, DCM, r.t., 0.5 h) followed by desilylation and neutralization gave 32 (85%). The synthesis of the sialylated trisaccharide 33 ( Figure S43, S44) has been recently described [32].

Isothermal Titration Calorimetry
Binding affinities (Kd values) and stoichiometry (n value) of hGal-3 for methyl lactoside (1) and some strategically designed analogs of lactose (4, 6, 11 and 12) as well as Lac-NAc (20, 23, 27, 30, 32, 33) were measured by isothermal titration calorimetry (ITC). All the analogs showed "n" values close to 1.0, indicating that they bound to a single binding site on the hGal-3 monomer ( Figure 1). When interacted with Gal-3, all the analogs tested in this study showed higher affinities compared to methyl lactoside (1). The Kd value of
Removal of 3 -O-sulfate group from lactoside analogs (as in 4 and 6) and LacNAc analogs (as in 27 and 32) reduced their affinity for hGal-3. The Kd values of 4 and 6 were 47.6 µM and 50.0 µM, respectively. Analogs 27 and 32 showed Kd values of 38.0 µM and 52.6 µM, respectively. These results suggest that the binding site of hGal-3 can beneficially accommodate an anionic sulfate group. This observation is consistent with a report that hGal-3 recognizes sulfated glycosaminoglycans (GAGs) [11]. Moreover, the anionic characters of the 3 -O-sulfated analogs in both series can clearly compensate for the cationic environment of the hGal-3 binding site near the 3 -position (see X-Ray analysis below).
Unlike galectin-1 (Gal-1), hGal-3 can tolerate terminal sialic acid of a glycan chain. In the present study, when the propargylated LacNAc (27) contained a terminal sialic acid residue (as in analog 33), the affinity was slightly reduced (compared to 27), but hGal-3 could still recognize the sialylated analog 33. The -T∆S values (Table 1) suggest that entropy played small but favorable roles in the binding of all the analogs, except 20, 32 and 33, where enthalpies were relatively dominant and the entropies were unfavorable. Entropic contributions were comparatively higher in the binding of 6, 11, and 23. This observation is in line with previously published data concerning the important role of conformational entropy and solvation entropy in protein-ligand binding [22] (see difference between 4 and 6). Overall, the ITC data showed that the binding site hGal-3 can accommodate different variations of the parent lactosyl moiety and that strategic modifications of lactose and LacNAc can increase their binding affinity for hGal-3.
the present study, when the propargylated LacNAc (27) contained a terminal sialic residue (as in analog 33), the affinity was slightly reduced (compared to 27), but hG could still recognize the sialylated analog 33. The -TS values (Table 1) suggest tha tropy played small but favorable roles in the binding of all the analogs, except 20, 32 33, where enthalpies were relatively dominant and the entropies were unfavorable tropic contributions were comparatively higher in the binding of 6, 11, and 23. This o vation is in line with previously published data concerning the important role of co mational entropy and solvation entropy in protein-ligand binding [22] (see differenc tween 4 and 6). Overall, the ITC data showed that the binding site hGal-3 can accom date different variations of the parent lactosyl moiety and that strategic modificatio lactose and LacNAc can increase their binding affinity for hGal-3.

Structure of Gal-3C in Complex with 23
This is the first structure to be reported of a sulfated sugar bound to the active site of the Gal-3 CRD. The molecular structure of the sugar, 23, used in this study is shown in Figure 2.

Structure of Gal-3C in Complex with 23
This is the first structure to be reported of a sulfated sugar bound to the active site of the Gal-3 CRD. The molecular structure of the sugar, 23, used in this study is shown in Figure 2.

Structure of Gal-3C in Complex with 23
This is the first structure to be reported of a sulfated sugar bound to the active site of the Gal-3 CRD. The molecular structure of the sugar, 23, used in this study is shown in Figure 2.

Structure of Gal-3C in Complex with 23
This is the first structure to be reported of a sulfated sugar bound to the active site of the Gal-3 CRD. The molecular structure of the sugar, 23, used in this study is shown in Figure 2.

Structure of Gal-3C in Complex with 23
This is the first structure to be reported of a sulfated sugar bound to the active site of the Gal-3 CRD. The molecular structure of the sugar, 23, used in this study is shown in Figure 2.

Structure of Gal-3C in Complex with 23
This is the first structure to be reported of a sulfated sugar bound to the active site of the Gal-3 CRD. The molecular structure of the sugar, 23, used in this study is shown in Figure 2.

Structure of Gal-3C in Complex with 23
This is the first structure to be reported of a sulfated sugar bound to the active site of the Gal-3 CRD. The molecular structure of the sugar, 23, used in this study is shown in Figure 2.

Structure of Gal-3C in Complex with 23
This is the first structure to be reported of a sulfated sugar bound to the active site of the Gal-3 CRD. The molecular structure of the sugar, 23, used in this study is shown in Figure 2.

Structure of Gal-3C in Complex with 23
This is the first structure to be reported of a sulfated sugar bound to the active site of the Gal-3 CRD. The molecular structure of the sugar, 23, used in this study is shown in Figure 2. Compound 23 is bound to the active site of the CRD in a very similar manne lactose and LacNAc (Figure 2a). Residues His158, Asn160, Arg162, Asn174, Trp181 Glu184 are involved in direct contacts, and residues Arg144, Asp148 and Glu165 m contact through waters. Figure 2a shows the electron density map for the sugar bound the Gal-3 CRD active site. There is very good electron density for the sulfate and the sugar moieties, while the nitro-phenyl substituent at O1′ does not show nearly any e tron density. This is reflected on the B factor distribution of the molecule 23 ( Figure  that shows the galactopyranose moiety firmly anchored to the active site with an aver B factor of 17.42 Å 2 . The sulfate group shows a higher average B factor of 32.77 Å 2 , refl Compound 23 is bound to the active site of the CRD in a very similar manner as lactose and LacNAc (Figure 2a). Residues His158, Asn160, Arg162, Asn174, Trp181 and Glu184 are involved in direct contacts, and residues Arg144, Asp148 and Glu165 make contact through waters. Figure 2a shows the electron density map for the sugar bound to the Gal-3 CRD active site. There is very good electron density for the sulfate and the two sugar moieties, while the nitro-phenyl substituent at O1 does not show nearly any electron density. This is reflected on the B factor distribution of the molecule 23 (Figure 2b) that shows the galactopyranose moiety firmly anchored to the active site with an average B factor of 17.42 Å 2 . The sulfate group shows a higher average B factor of 32.77 Å 2 , reflecting no direct contact with the protein. Meanwhile, the N-acetyl glucopyranose shows a more relaxed binding with an average B factor of 52.58 Å 2 . Finally, the nitro-phenyl substituent does not have any contact with protein residues and shows a very high B factor average of 178.97 Å 2 .
The typical interactions of the galactopyranose and the glucopyranose moieties of 23 are very similar to those of lactose. The sulfate group contacts the protein through water molecules; one oxygen interacts with one water (S1) that is in contact with Trp181, and a second oxygen contacts two waters (S2 and S3) that are in contact with Asp148 ( Figure 3a). Also, the oxygen of the N-acetyl substituent in the glucopyranose moiety makes one direct contact with Glu184. The superposition of all Gal-3 CRD structures present in the PDB in complex with lactose, LacNAc (β-D-galactopyranose-(1-4)-2-acetamido-2-deoxy-β-D-glucopyranose) (PDB ID 1KJL) [33] and LacNAcNAc (β-D-galactopyranose-(1-4)-N-acetyl-2-(acetylamino)-2-deoxy-β-D-glucopyranosylamine) (PDB ID 5NF7) [34,35], is shown in Figure 3b. Most of the residues in the binding site are in very similar positions; only Arg144 shows different conformations. Moreover, the conserved water network adding to the binding of sugar can be observed in Figure 3b. Seven waters are conserved in our structure (S1 through S7), water 8 (S8) is missing in our structure, and one water (S9) has been replaced by one of the oxygens from the sulfate group.
The superposition of all Gal-3 CRD structures with a sugar containing a N-acetyl substituent at O2′ are shown in Figure 3c. There are only three structures in the PDB, two in complex with LacNAc and one in complex with LacNAcNAc. The position of this group is not very well fixed. In two of the structures the oxygen does not appear to contact Glu184. One structure makes a water (S10)-mediated contact with this latter residue. In our structure we can observe a direct contact between the oxygen of the N-acetyl group and Glu184 (dashed line in Figure 3c). The superposition of all Gal-3 CRD structures present in the PDB in complex with lactose, LacNAc (β-D-galactopyranose-(1-4)-2-acetamido-2-deoxy-β-D-glucopyranose) (PDB ID 1KJL) [33] and LacNAcNAc (β-D-galactopyranose-(1-4)-N-acetyl-2-(acetyla-mino)-2deoxy-β-D-glucopyranosylamine) (PDB ID 5NF7) [34,35], is shown in Figure 3b. Most of the residues in the binding site are in very similar positions; only Arg144 shows different conformations. Moreover, the conserved water network adding to the binding of sugar can be observed in Figure 3b. Seven waters are conserved in our structure (S1 through S7), water 8 (S8) is missing in our structure, and one water (S9) has been replaced by one of the oxygens from the sulfate group.
The superposition of all Gal-3 CRD structures with a sugar containing a N-acetyl substituent at O2 are shown in Figure 3c. There are only three structures in the PDB, two in complex with LacNAc and one in complex with LacNAcNAc. The position of this group is not very well fixed. In two of the structures the oxygen does not appear to contact Glu184. One structure makes a water (S10)-mediated contact with this latter residue. In our structure we can observe a direct contact between the oxygen of the N-acetyl group and Glu184 (dashed line in Figure 3c).

General Synthetic Methods
All reactions in organic medium were performed in standard oven-dried glassware under an inert atmosphere of nitrogen using freshly distilled solvents. Solvents and reagents were deoxygenated, when necessary, by purging with nitrogen. All reagents were used as supplied without prior purification, unless otherwise stated, and obtained from Sigma-Aldrich Chemical Co. Ltd.

General Synthetic Procedure A: Preparation of 3 -O-Sulfated Lactosides
A mixture of deacetylated lactosides (1 eq.) and dibutyltin oxide (Bu 2 SnO, 1.08 eq.) in MeOH (4 mL per 0.1 mmol of the lactoside) was stirred at 80 • C for 4 h under nitrogen atmosphere. The solution was then concentrated, and sulfur trioxide-triethylamine complex (Et 3 N.SO 3 ) (1.2 eq.) and dry DMF (4 mL per 0.1 mmol of the lactoside) were added. After stirring at 60 • C for 17 h, the reaction was quenched with methanol and the reaction mixture was concentrated in vacuo. The residue was purified through a classical column chromatography to give desired compound.

General Synthetic Procedure B: Zemplén Transesterification Reaction
To a solution of lactoside (1 eq.) in dry methanol (2 mL per 0.1 mmol of the lactoside) was added a solution of sodium methoxide (25% in MeOH, 0.5 eq.). After stirring at room temperature for 3 h, the basic media was neutralized by addition of ion-exchange resin (Amberlite IR 120 H + ). The reaction mixture was filtered through a pad of celite and concentrated in vacuo to afford the de-O-acetylated lactosides.

General Synthetic Procedure C: Protection of Primary Alcohol with Tert-Butyldiphenylsilyl Ether (TBDPS)
To a solution of lactoside (1 eq.) in pyridine (2 mL per 0.1 mmol of lactoside) was added TBDPSCl (1.5 eq. per primary alcohol) at room temperature under nitrogen atmosphere. After 8 h of stirring the reaction was quenched with methanol, the reaction mixture was co-evaporated with toluene under vacuum, and the residue was purified using column chromatography.

Protein Expression and Purification
The vector pET41-Gal-3C was transformed into E. coli Tuner (DE3) cells (Novagen, Merck, Madrid, Spain). These cells were grown in 2xYT medium supplemented with kanamycin (30 mg L −1 ) at 37 • C; when the cells attained an OD 600 of 0.6-0.8 the temperature was then dropped to 20 • C. When the temperature stabilized (approx. 15 min) the expression of the protein was induced by the addition of 0.1 mM isopropyl-β-Dthiogalactopyranoside (IPTG), and let to grow for an additional 16 h. The cells were harvested by centrifugation and resuspended in the lysis buffer containing 10 mM TRIS-HCl at pH 8.0 and 1% (v/v) Triton X-100 (Sigma-Aldrich, Merck, Madrid, Spain). The cells were lysed by sonication, and the insoluble fraction was removed by centrifugation at 45,000× g for 1 h; the supernatant was then loaded onto a lactosylated Sepharose 4B column. The column was washed extensively with PBS buffer containing 2 mM β-mercaptoethanol (β-ME). The protein was eluted from the column in PBS buffer containing 50 mM lactose and 2 mM β-ME. The fractions containing the protein were pooled and dialyzed against 1 L of PBS buffer containing 2 mM β-ME with four changes to remove the lactose. Crystals were harvested, for data collection, in 0.1 M TRIS-HCl at pH 8.5 containing 36% PEG 6000, which is a cryo-solution, and flash-cooled in liquid nitrogen. X-Ray data collection experiments were performed at the ALBA Synchrotron (Cerdanyola del Vallès, Spain) BL13 XALOC beamline. Data were indexed and integrated, scaled and merged using the software AutoProc (https://www.globalphasing.com/autoproc/) [36] and Staraniso (https://staraniso.globalphasing.org/cgi-bin/staraniso.cgi) [37] from Globalphasing using XDS [38] and the programs POINTLESS (https://www.ccp4.ac.uk/html/pointless. html) [39], AIMLESS (https://www.ccp4.ac.uk/html/aimless.html) [40] from the CCP4 suite (https://www.ccp4.ac.uk/) [41].

Structure Determination
The structure was solved by molecular replacement using the CRD of the previously reported Gal-3 structure [42] (PDB: 6FOF) with Phaser [43]. The initial model was first refined using Phenix [44] and alternating manual building with Coot [45]. The final model was obtained by repetitive cycles of refinement; solvent molecules were added automatically and inspected visually for chemically plausible positions. The inhibitor molecule was added manually. The stereochemical quality of the model was assessed with MolProbity [46]. The structural figures were generated using the Pymol program (http://www.pymol.org). Data processing and refinement statistics are listed in Table 2.

Conclusions
This work described the syntheses of three series of lactose and N-acetyl-lactosaminebased analogs cooperatively modified at three key positions previously known to independently improve binding to human galectin-3 (hGal-3). ITC experiments showed that the best ligand belonged to the LacNAc family with an anionic sulfate group installed at the 3 -position. Interestingly, the nature of the aglycon had little influence on the overall affinities within the Lac and LacNAc series, an observation also corroborated by the X-ray data which showed the aglycon protruding outside the active CRD-binding pocket of hGal-3. The trisaccharide containing the sialic acid moiety at the 3 -position of the Lac-NAc analog can also be accommodated within the active site but did not show further improvement in binding affinity. The three best candidates all contained a 3 -sulfate group. Altogether, our data substantiate the important role of 3'-sulfated LacNAc as powerful antagonists for hGal-3. These results are relevant in the context of the search for more selective and effective inhibitors of Gal-3 in the treatment of cancer, inflammation and fibrosis. These newly designed inhibitors should be compared with other small molecule inhibitors, large polysaccharide antagonists, peptidomimetics and biological agents using in vitro and in vivo biological assays. Ongoing work is being performed on Gal-1 to further explore if selectivity amongst the galectin families is effective in the selection of improved ligands.